Method for producing and cleaving a fusion proteins with an n-terminal chymosin pro-peptide

ABSTRACT

An improved method for recovering recombinantly produced polypeptides is described. The method involves expressing the recombinant polypeptide as a fusion protein with a pro-peptide. The pro-peptide-polypeptide fusion protein can be cleaved and the recombinant polypeptide released under the appropriate conditions.

This application claims the benefit under 35 USC §119(e) from U.S.provisional application No. 60/044,254 filed Apr. 25, 1997, nowabandoned, which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to an improved method for recoveringrecombinantly produced polypeptides. The method involves expressing therecombinant polypeptide as a fusion protein with a pro-peptide. Thepro-peptide-polypeptide fusion protein can be cleaved and therecombinant protein released under the appropriate conditions.

BACKGROUND OF THE INVENTION

The preparation of valuable recombinant (genetically engineered)polypeptides, for example pharmaceutical proteins, relies frequently ontechniques which involve the production of these polypeptides as fusionor hybrid proteins. These techniques are based upon the preparation ofhybrid genes, i.e. genes comprising genetic material encoding thepolypeptide of interest linked to genetic material additional to thegene of interest. Production of the fusion polypeptide involves theintroduction of the hybrid gene into a biological host cell system, forexample yeast cells, which permits the expression and accumulation ofthe fusion polypeptide. Recovery of the polypeptide of interest involvesthe performance of a cleavage reaction which results in the separationof the desired polypeptide from the “fusion partner”.

Despite the additional steps which are required to produce a protein ofinterest as a fusion protein, rather than directly in its active form,the production of hybrid proteins has been found to overcome a number ofproblems. Firstly, overproduced polypeptides can aggregate in the hostcell in insoluble fractions known as inclusion bodies. Conversion ofthis insoluble material involves often slow and complex refoldingmethods, making protein purification difficult. Secondly, those proteinswhich are present in soluble form in the cytoplasm often are subject todegradation by host specific enzymes, thus reducing the amounts ofactive protein that can be recovered. Linking the polypeptide ofinterest to a fusion partner has been found to limit these problems.Fusion partners known to the prior art include maltose binding protein(Di Guan et al. (1988) Gene 67: 21-30), glutathione-S-transferase(Johnson (1989) Nature 338: 585-587), ubiquitin (Miller et al. (1989)Biotechnology 7: 698-704), β-galactosidase (Goeddel et al. (1979) Proc.Natl. Acad. Sci. (USA) 76: 106-110), and thioredoxin (LaVallie et al.(1993) Biotechnology 11:187-193).

It has also been proposed to employ fusion partners as affinitypeptides. This methodology facilitates the isolation and recovery of thefusion peptide from the host cells by exploiting the physico-chemicalproperties of the fusion partner. (See, for example, WO 91/11454).

Finally, the use of a fusion partner may enable the production of apeptide which would otherwise be too small to accumulate and recoverefficiently from a recombinant host cell system. This technology isdescribed, for example, by Schultz et al., (1987, J. Bacteriol. 169:5385-5392)

All of these procedures result in the production a hybrid protein inwhich the protein of interest is linked to an additional polypeptide. Inorder to recover the active polypeptide it is, in general, necessary toseparate the fusion partner from the polypeptide of interest. Mostcommonly, a cleavage reaction, either by enzymatic or by chemical means,is performed. Such reactions employ agents that act by hydrolysis ofpeptide bonds and the specificity of the cleavage agent is determined bythe identity of the amino acid residue at or near the peptide bond whichis cleaved.

Enzymes known to the prior art as “proteolytic enzymes” have been foundto be particularly well suited for the cleavage of fusion proteins. Thecleavage reaction is performed by contacting the fusion protein with aproteolytic enzyme under appropriate conditions. An example of thismethodology is described in U.S. Pat. No. 4,743,679 which discloses aprocess for the production of human epidermal growth factor comprisingcleavage of a fusion protein by Staphylococcus aureus V8 protease.

By contrast, chemical cleavage involves the use of chemical agents whichare known to permit hydrolysis of peptide bonds under specificconditions. Cyanogenbromide, for example, is known to cleave thepolypeptide chain at a methionine residue. A hydrolysis reaction for thecyanogenbromide cleavage of the proteins urease and phosphorylase bbased on this technique is described by Sekita et al. ((1975), Keio J.Med. 24: 203-210).

Both chemical and enzymatic cleavage reactions require the presence of apeptide bond which can be cleaved by the cleavage agent which isemployed. For this reason it is often desirable to place an appropriatetarget sequence at the junction of the fusion partner and the targetprotein. Fusion peptides comprising “linker” sequences containing atarget for a proteolytic enzyme may readily be constructed usingconventional art-recognized genetic engineering techniques.

Despite their great utility, the prior art cleavage methods have beenrecognized to be either inefficient or lack cleavage specificity.Inefficient cleavage results in low protein purification efficiency,while the lack of cleavage specificity results in cleavage at severallocations resulting in product loss and generation of contaminatingfragments. This results frequently in the recovery of only a smallfraction of the desired protein. In addition, the currently widely usedproteolytic enzymes, such as blood clotting factor Xa and thrombin, areexpensive, and contamination of final product with blood pathogens is aconsideration.

In view of these shortcomings, the limitations of the cleavage methodsknown to the prior art are apparent.

Zymogens, such as pepsin and chymosin, are enzymes which are synthesizedas inactive precursors in vivo. Under appropriate conditions, zymogensare activated to form the mature active protein in a process involvingthe cleavage of an amino-terminal peptide which can be referred to asthe “pro-peptide”, “pro-region” or “pro-sequence”. Activation ofzymogens may require the presence of an additional specific proteolyticenzyme, for example various hormones, such as insulin, are processed bya specific proteolytic enzyme. Alternatively, activation may occurwithout an additional enzymatic catalyst. These kinds of zymogens arefrequently referred to as “autocatalytically maturing” zymogens.Examples of autocatalytically maturing zymogens include pepsin,pepsinogen and chymosin which are activated by an acidic environment,for example in the mammalian stomach.

The autocatalytic activation and processing of zymogens has beendocumented extensively (see for example, McCaman and Cummings, (1986),J. Biol. Chem. 261: 15345-15348; Koelsch et al. (1994). FEBS Letters343: 6-10). It has also been documented that activation of the zymogendoes not necessarily require a physical linkage of the pro-peptide tothe mature protein (Silen et al. (1989), Nature, 341: 462-464).

There is a need for an improved process for recovering recombinantlyproduced polypeptides from their expression systems.

SUMMARY OF THE INVENTION

The present inventors have developed a novel method for recoveringrecombinantly produced polypeptides. The method involves expressing thepolypeptide as a fusion protein with a pro-peptide so that therecombinant polypeptide can be cleaved from the pro-peptide under theappropriate conditions.

In one aspect, the invention provides a chimeric nucleic acid sequenceencoding a fusion protein, the chimeric nucleic acid sequence comprisinga first nucleic acid sequence encoding a pro-peptide derived from anautocatalytically maturing zymogen and a second nucleic acid sequenceencoding a polypeptide that is heterologous to the pro-peptide.

In another aspect the present invention provides a fusion proteincomprising (a) a pro-peptide derived from an autocatalytically maturingzymogen and (b) a polypeptide that is heterologous to the pro-peptide.In one embodiment, the heterologous polypeptide is a therapeutic ornutritional peptide and the fusion protein may be administered as apharmaceutical or food composition. In such an embodiment theheterologous polypeptide may be cleaved once the composition isdelivered to the host as a result of the physiological conditions at thetarget organ, tissue or in the bodily fluid.

In a further aspect, the present invention provides a method for thepreparation of a recombinant polypeptide comprising

-   (a) introducing into a host cell an expression vector comprising:    -   (1) a nucleic acid sequence capable of regulating transcription        in a host cell, operatively linked to    -   (2) a chimeric nucleic acid sequence encoding a fusion protein,        the chimeric nucleic acid sequence comprising (a) a nucleic acid        sequence encoding a pro-peptide derived from an        autocatalytically maturing zymogen, linked in reading frame        to (b) a nucleic acid sequence heterologous to the pro-peptide        and encoding the recombinant polypeptide; operatively linked to    -   (3) a nucleic acid sequence encoding a termination region        functional in the host cell,-   (b) growing the host cell to produce said fusion protein; and-   (c) altering the environment of the fusion protein so that the    pro-peptide is cleaved from the fusion protein to release the    recombinant polypeptide.

The environment of the fusion protein can be altered using many meansincluding altering the pH, temperature or salt concentration or otheralterations that permit to pro-peptide to self-cleave from the fusionprotein to release to recombinant polypeptide. In a preferredembodiment, the mature zymogen is added to the method in step (c) toassist in the cleavage of the propeptide from the fusion protein.

Other features and advantages of the present invention will becomereadily apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art of this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIG. 1 is the nucleic acid (SEQ.ID.NO.:1) and deduced amino acidsequence (SEQ.ID.NO.:2) of a GST-Chymosin pro-peptide-Hirudin sequence.

FIG. 2 is the nucleic acid (SEQ.ID.NO.:3) and deduced amino acidsequence (SEQ.ID.NO.:4) of a poly histidine tagged chymosin pro-peptidecarp growth hormone (His-Pro-cGH) fusion protein.

FIG. 3 is a schematic diagram of the Pro-cGH fusion construct.

FIG. 4 illustrates the in vitro cleavage of purified His-Pro-cGH.

FIG. 5 illustrates the in vivo cleavage of purified His-Pro-cGH.

DETAILED DESCRIPTION OF THE INVENTION

As hereinbefore mentioned, the present invention relates to a novelmethod for preparing and recovering recombinant polypeptides, chimericnucleic acid sequences encoding fusion proteins and fusion proteinsuseful in pharmaceutical and nutritional compositions.

Accordingly, the present invention provides a method for the preparationof a recombinant polypeptide comprising:

-   (a) introducing into a host cell an expression vector comprising:    -   (1) a nucleic acid sequence capable of regulating transcription        in a host cell, operatively linked to    -   (2) a chimeric nucleic acid sequence encoding a fusion protein,        the chimeric nucleic acid sequence comprising (a) a nucleic acid        sequence encoding a pro-peptide derived from an        autocatalytically maturing zymogen, linked in reading frame        to (b) a nucleic acid sequence heterologous to the pro-peptide        and encoding the recombinant polypeptide, operatively linked to    -   (3) a nucleic acid sequence encoding a termination region        functional in said host cell,-   b) growing the host cell to produce said fusion protein; and-   c) altering the environment of the fusion protein so that the    pro-peptide is cleaved from the fusion protein to release the    recombinant polypeptide.

The environment of the fusion protein can be altered using many meansincluding altering the pH, temperature or salt concentration or otheralterations that permit to pro-peptide to self-cleave from the fusionprotein to release to recombinant polypeptide. In a preferredembodiment, the mature zymogen is added to the method in step (c) toassist in the cleavage of the propeptide from the fusion protein

The term “pro-peptide” as used herein means the amino terminal portionof a zymogen or a functional portion thereof up to the maturation site.

The term “autocatalytically maturing zymogen” as used herein means that:(i) the zymogen can be processed to its active form without requiring anadditional specific protease and that (ii) the mature form of thezymogen can assist in the cleavage reaction.

The term “mature zymogen” as used herein means a zymogen that does notcontain the pro-peptide sequence or portion.

The polypeptide can be any polypeptide that is heterologous to thepro-peptide, meaning that it is not the mature protein that is normallyassociated with the pro-peptide as a zymogen.

In another aspect, the invention provides a chimeric nucleic acidsequence encoding a fusion protein, the chimeric nucleic acid sequencecomprising a first nucleic acid sequence encoding a pro-peptide derivedfrom an autocatalytically maturing zymogen and a second nucleic acidsequence encoding a polypeptide that is heterologous to the pro-peptide.The chimeric nucleic acid sequence generally does not include a nucleicacid sequence encoding the entire zymogen.

The chimeric nucleic acid sequences which encode the fusion proteins ofthe present invention can be incorporated in a known manner into arecombinant expression vector which ensures good expression in a hostcell.

Accordingly, the present invention also includes a recombinantexpression vector comprising a chimeric nucleic acid molecule of thepresent invention operatively linked to a regulatory sequence andtermination region suitable for expression in a host cell.

The term “nucleic acid sequence” refers to a sequence of nucleotide ornucleoside monomers consisting of naturally occurring bases, sugars, andintersugar (backbone) linkages. The term also includes modified orsubstituted sequences comprising non-naturally occurring monomers orportions thereof, which function similarly. The nucleic acid sequencesof the present invention may be ribonucleic (RNA) or deoxyribonucleicacids (DNA) and may contain naturally occurring bases including adenine,guanine, cytosine, thymidine and uracil. The sequences may also containmodified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl,2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-azauracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil,8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines,8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines,8-amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxylguanine and other 8-substituted guanines, other aza and deaza uracils,thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uraciland 5-trifluoro cytosine.

The term “suitable for expression in a host cell” means that therecombinant expression vectors contain the chimeric nucleic acidsequence of the invention, a regulatory sequence and a terminationregion, selected on the basis of the host cells to be used forexpression, which is operatively linked to the chimeric nucleic acidsequence. Operatively linked is intended to mean that the chimericnucleic acid sequence is linked to a regulatory sequence and atermination region in a manner which allows expression of the chimericsequence. Regulatory sequences and termination regions areart-recognized and are selected to direct expression of the desiredprotein in an appropriate host cell. Accordingly, the term regulatorysequence includes promoters, enhancers and other expression controlelements. Such regulatory sequences are known to those skilled in theart or one described in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990) can be used. Itshould be understood that the design of the expression vector may dependon such factors as the choice of the host cell to be transformed and/orthe type of protein desired to be expressed. Such expression vectors canbe used to transform cells to thereby produce fusion proteins orpeptides encoded by nucleic acids as described herein.

The recombinant expression vectors of the invention can be designed forexpression of encoded fusion proteins in prokaryotic or eukaryoticcells. For example, fusion proteins can be expressed in bacterial cellssuch as E. coli, insect cells (using, for example baculovirus), yeastcells, plant cells or mammalian cells. Other suitable host cells can befound in Goeddel, Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990). The type of host cell which isselected to express the fusion protein is not critical to the presentinvention and may be as desired.

Expression in prokaryotes is most often carried out in E. coli withvectors containing constitutive or inducible promoters directing theexpression of the fusion proteins. Inducible expression vectors includepTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al.,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990) 60-89). While target gene expression relies onhost RNA polymerase transcription from the hybrid trp-lac fusionpromoter in pTrc, expression of target genes inserted into pET 11drelies on transcription from the T7 gn10-lac 0 fusion promoter mediatedby coexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 under the transcriptional control of thelacUV 5 promoter. Another attractive bacterial expression system is thepGEX expression system (Pharmacia) in which genes are expressed asfusion products of glutathione-S-transferase (GST), allowing easypurification of the expressed gene from a GST affinity column.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in host bacteria with an impaired capacity toproteolytically cleave the recombinantly expressed proteins (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Another strategy is to alterthe nucleic acid sequence of the chimeric DNA to be inserted into anexpression vector so that the individual codons for each amino acidwould be those preferentially utilized in highly expressed E. coliproteins (Wada et al., (1992) Nuc. Acids Res. 20: 2111-2118). Suchalteration of nucleic acid sequences of the invention could be carriedout by standard DNA synthesis techniques.

Examples of vectors for expression in yeast S. cereviseae includepYepSec1 (Baldari. et al., (1987) Embo J. 6:229-234), pMFa (Kurjan andHerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.).

Baculovirus vectors available for expression of proteins in culturedinsect cells (SF 9 cells) include the pAc series (Smith et al., (1983)Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow, V. A., andSummers, M. D., (1989) Virology).

Vectors such as the Ti and Ri plasmids are available for transformationand expression of plants. These vectors specify DNA transfer functionsand are used when it is desired that the constructs are introduced intothe plant and stably integrated into the genome viaAgrobacterium-mediated transformation.

A typical construct consists, in the 5′ to 3′ direction, of a regulatoryregion complete with a promoter capable of directing expression inplant, a protein coding region, and a sequence containing atranscriptional termination signal functional in plants. The sequencescomprising the construct may be either natural or synthetic or anycombination thereof.

Both non-seed specific promoters, such as the 35-S CaMV promoter(Rothstein et al., (1987), Gene 53: 153-161) and, if seed specificexpression is desired, seed-specific promoters such as the phaseolinpromoter (Sengupta-Gopalan et al., (1985), PNAS USA 82: 3320-3324) orthe Arabidopsis 18 kDa oleosin (Van Rooijen et al., (1992) Plant Mol.Biol. 18: 1177-1179) promoters may be used. In addition to the promoter,the regulatory region contains a ribosome binding site enablingtranslation of the transcripts in plants and may also contain one ormore enhancer sequences, such as the AMV leader (Jobling and Gehrke,(1987), Nature 325: 622-625), to increase the expression of product.

The coding region of the construct will typically be comprised ofsequences encoding a pro-peptide region fused in frame to a desiredprotein and ending with a translational termination codon. The sequencemay also include introns.

The region containing the transcriptional termination signal maycomprise any such sequence functional in plants such as the nopalinesynthase termination sequence and additionally may include enhancersequences to increase the expression of product.

The various components of the construct are ligated together usingconventional methods, typically into a pUC-based vector. This constructmay then be introduced into an Agrobacterium vector and subsequentlyinto host plants, using one of the transformation procedures outlinedbelow.

The expression vectors will normally also contain a marker which enablesexpression in plant cells. Conveniently, the marker may be a resistanceto a herbicide, for example glyphosate, or an antibiotic, such askanamycin, G418, bleomycin, hygromycin, chloramphenicol or the like. Theparticular marker employed will be one which will permit selection oftransformed cells from cells lacking the introduced recombinant nucleicacid molecule.

A variety of techniques is available for the introduction of nucleicacid sequences, in particular DNA into plant host cells. For example,the chimeric DNA constructs may be introduced into host cells obtainedfrom dicotyledonous plants, such as tobacco, and oleaginous species,such as B. napus using standard Agrobacterium vectors; by atransformation protocol such as that described by Moloney et al.,(1989), (Plant Cell Rep., 8: 238-242) or Hinchee et al., (1988),(Bio/Technol., 6: 915-922); or other techniques known to those skilledin the art. For example, the use of T-DNA for transformation of plantcells has received extensive study and is amply described in EPA SerialNo. 120,516; Hoekema et al., (1985), (Chapter V, In: The Binary PlantVector System Offset-drukkerij Kanters B. V., Alblasserdam); Knauf, etal., (1983), (Genetic Analysis of Host Range Expression byAgrobacterium, p. 245, In Molecular Genetics of the Bacteria-PlantInteraction, Puhler, A. ed., Springer-Verlag, NY); and An et al.,(1985), (EMBO J., 4: 277-284). Conveniently, explants may be cultivatedwith A. tumefaciens or A. rhizogenes to allow for transfer of thetranscription construct to the plant cells. Following transformationusing Agrobacterium the plant cells are dispersed in an appropriatemedium for selection, subsequently callus, shoots and eventuallyplantlets are recovered. The Agrobacterium host will harbour a plasmidcomprising the vir genes necessary for transfer of the T-DNA to theplant cells. For injection and electroporation, (see below) disarmedTi-plasmids (lacking the tumour genes, particularly the T-DNA region)may be introduced into the plant cell.

The use of non-Agrobacterium techniques permits the use of theconstructs described herein to obtain transformation and expression in awide variety of monocotyledonous and dicotyledonous plants and otherorganisms. These techniques are especially useful for species that areintractable in an Agrobacterium transformation system. Other techniquesfor gene transfer include biolistics (Sanford, (1988), Trends inBiotech., 6: 299-302), electroporation (Fromm et al., (1985), Proc.Natl. Acad. Sci. USA, 82: 5824-5828; Riggs and Bates, (1986), Proc.Natl. Acad. Sci. USA 83: 5602-5606) or PEG-mediated DNA uptake (Potrykuset al., (1985), Mol. Gen. Genet., 199: 169-177).

In a specific application, such as to B. napus, the host cells targetedto receive recombinant DNA constructs typically will be derived fromcotyledonary petioles as described by Moloney et al., (1989, Plant CellRep., 8: 238-242). Other examples using commercial oil seeds includecotyledon transformation in soybean explants (Hinchee et al., (1988).Bio/Technology, 6: 915-922) and stem transformation of cotton (Umbeck etal., (1981), Bio/Technology, 5: 263-266).

Following transformation, the cells, for example as leaf discs, aregrown in selective medium. Once shoots begin to emerge, they are excisedand placed onto rooting medium. After sufficient roots have formed, theplants are transferred to soil. Putative transformed plants are thentested for presence of a marker. Southern blotting is performed ongenomic DNA using an appropriate probe, for example a chymosinpro-sequence, to show that integration of the desired sequences into thehost cell genome has occurred.

Transformed plants grown in accordance with conventional ways, areallowed to set seed. See, for example, McCormick et al. (1986, PlantCell Reports, 5: 81-84). Northern blotting can be carried out using anappropriate gene probe with RNA isolated from tissue in whichtranscription is expected to occur, such as a seed embryo. The size ofthe transcripts can then be compared with the predicted size for thefusion protein transcript.

Two or more generations of transgenic plants may be grown and eithercrossed or selfed to allow identification of plants and strains withdesired phenotypic characteristics including production of recombinantproteins. It may be desirable to ensure homozygosity of the plants,strains or lines producing recombinant proteins to assure continuedinheritance of the recombinant trait. Methods of selecting homozygousplants are well know to those skilled in the art of plant breeding andinclude recurrent selfing and selection and anther and microsporeculture. Homozygous plants may also be obtained by transformation ofhaploid cells or tissues followed by regeneration of haploid plantletssubsequently converted to diploid plants by any number of known means,(e.g.: treatment with colchicine or other microtubule disruptingagents).

The polypeptide of the present invention may be any polypeptide that isnot normally fused to the pro-peptide used in the method. Thepolypeptide is preferentially stable under cleavage conditions, forexample at acidic pH, and the polypeptide may be activated aftercleavage upon adjusting the pH, or altering the environment otherwise sothat conditions optimal for enzymatic activity are generated. Thecleavage reaction may be performed any time upon commencement of theproduction of the fusion protein in a recombinant cell system. Inpreferred embodiments the cleavage reaction is performed using crudecellular extracts producing the recombinant protein or any purifiedfraction thereof.

The pro-peptide used in the present invention may be any pro-peptidederived from any autocatalytically maturing zymogen, including thosepro-peptides derived from proteases, including aspartic proteases,serine proteases and cysteine proteases. In preferred embodiments of theinvention, the pro-peptide is derived from chymosin, pepsin, HIV-1protease, pepsinogen, cathepsin or yeast proteinase A. The amino acidand/or DNA sequences of pepsinogen (Ong et al. (1968), J. Biol. Chem.6104-6109; Pedersen et al., (1973), FEBS Letters, 35: 255-526), chymosin(Foltmann et al., (1977); Harris et al., (1982), Nucl. Acids. Res., 10:2177-2187), yeast proteinase A (Ammerer et al., (1986), Mol. Cell. Biol.6: 2490-2499; Woolford et al., (1986), Mol. Cell. Biol. 6: 2500-2510),HIV-1 protease (Ratner et al., (1987), AIDS Res. Human Retrovir. 3:57-69.), cathepsin (McIntyre et al., (1994), J. Biol. Chem. 269:567-572) and pepsin are available (Koelsch et al. (1994), FEBS Lett.343: 6-10). Based on these sequences cDNA clones comprising the geneticmaterial coding for the pro-peptides may be prepared and fusion genesmay be prepared in accordance with the present invention and practisingtechniques commonly known to those skilled in the art (see e.g. Sambrooket al. (1990), Molecular Cloning, 2nd Ed., Cold Spring Harbor Press).

To identify other pro-sequences having the desired characteristics,where a zymogen undergoing autocatalytic cleavage has been isolated (forexample chymosin and yeast protein A), the protein may be partiallysequenced, so that a nucleic acid probe may be designed to identifyother pro-peptides. The nucleic acid probe may be used to screen cDNA orgenomic libraries prepared from any living cell or virus. Sequenceswhich hybridize with the probe under stringent conditions may then beisolated.

Other pro-sequences may also be isolated by screening of cDNA expressionlibraries. Antibodies against existing pro-peptides may be obtained andcDNA expression libraries may be screened with these antibodiesessentially as described by Huynh et al. (1985, in DNA cloning, Vol. 1,a Practical Approach, ed. D. M. Glover, IRL Press). Expression librariesmay be prepared from any living cell or virus.

Other zymogens which are autocatalytically processed may be discoveredby those skilled in the art. The actual pro-sequence which is selectedis not of critical importance and may be as desired. It is to be clearlyunderstood that the pro-sequence of any autocatalytically maturingzymogen may be employed without departing from the spirit or scope ofthe present invention.

Upon isolation of a pro-sequence, the pro-peptide encoding geneticmaterial may be fused to the genetic material encoding polypeptide ofinterest using DNA cloning techniques known to skilled artisans such asrestriction digestion, ligation, gel electrophoresis, DNA sequencing andPCR. A wide variety of cloning vectors are available to perform thenecessary cloning steps. Especially suitable for this purpose are thecloning vectors which include a replication system that is functional inE. coli such as pBR322, the pUC series, M13mp series, pACYC184,pBluescript etc. Sequences may be introduced into these vectors and thevectors may be used to transform the E. coli host, which may be grown inan appropriate medium. Plasmids may be recovered from the cells uponharvesting and lysing the cells.

The invention also includes the full length pro-peptide as well asfunctional portions of the pro-peptide or functional mutated forms ofthe pro-peptide. Mutated forms of the pro-peptide may be used to obtainspecific cleavage between the pro-peptide and a heterologous protein.Mutations in the pro-peptide could alter the optimal conditions, such astemperature, pH and salt concentration, under which cleavage of aheterologous peptide is achieved (McCaman, M. T. and Cummings, D. B.,(1986), J. Biol. Chem. 261:15345-15348). Depending on the pro-peptide,cleavage of the heterologous protein from various pro-peptides, will beoptimal under varying different conditions. Thus the invention will beamenable to heterologous proteins which are preferentially cleaved undera variety of desirable conditions.

The nucleic acid sequence encoding the heterologous polypeptide may befused upstream or downstream of the nucleic acid sequence encoding thepro-peptide and concatamers containing repetitive units of thepro-peptide fused to the heterologous protein may be employed. Inpreferred embodiments, the heterologous protein is fused downstream ofthe pro-peptide. The nucleic acid sequence encoding the pro-peptidegenerally does not include the mature form of the zymogen.

In one embodiment, the pro-peptide is a pro-peptide derived fromchymosin and the heterologous polypeptide is hirudin (Dodt et al.,(1984), FEBS Letters 65:180-183). In particular, the present inventorshave constructed a chimeric DNA sequence in which the DNA encoding thechymosin pro-peptide was fused upstream of the DNA sequence encoding theleech anticoagulant protein hirudin. The gene fusion (Pro-Hirudin) wasexpressed in E. coli cells. It was found that upon lowering of the pH topH 2, and more preferably to pH 4.5, and in the presence of a smallquantity of mature chymosin, the heterologously fused protein, hirudin,was efficiently cleaved from the chymosin pro-peptide.

Autocatalytic cleavage requires an alteration of the environment of thefusion peptide. This may include alterations in pH, temperature, saltconcentrations, the concentrations of other chemical agents or any otheralteration resulting in environmental conditions that will permitautocatalytic cleavage of the fusion protein. The environment may bealtered by the delivery of the fusion protein into an appropriatecleavage environment. The cleavage environment may be a physiologicalenvironment, such as for example in the mammalian stomach, gut, kidneys,milk or blood, or the environmental conditions may be man-made. Thecleavage environment may also be generated by the addition of an agentor agents or by altering the temperature of the environment of thefusion protein. The cleavage reaction may take place when the fusionprotein is pure or substantially pure, as well as when it is present incruder preparations, such as cellular extracts.

In a preferred embodiment, the inventors have employed mature chymosinto assist in the cleavage reaction. Generally, the addition of themature enzyme will assist in the cleavage reaction. The enzyme used forthis reaction may be homologous to the pro-peptide, for example,chymosin may be used to assist cleavage of pro-chymosin fused to adesired protein, or heterologous to the pro-peptide, for example, pepsinmay be used to assist in cleavage of a pro-chymosin fused to a desiredprotein.

Although in a preferred embodiment mature chymosin is added, it isconceivable that the use of other pro-peptides may not require theaddition of the mature peptide in order to accomplish efficientcleavage.

Activation of the fusion protein may be in vitro or in vivo. In oneembodiment, the pro-peptide is used to facilitate cleavage from proteinsrecombinantly produced on oil bodies as disclosed in PCT applicationPublication No. WO 96/21029. In this embodiment, the pro-peptide wouldbe fused downstream of an oil body protein and upstream of therecombinant protein or peptide of interest.

In another in vivo application, two vectors would be introduced in thesame host. In one vector expression of the zymogen or the mature proteinwould be controlled by an inducible promoter system. The other vectorwould comprise a pro-peptide fused upstream of an heterologous proteinof interest. Thus it is possible to control the moment of cleavage ofthe peptide or protein downstream of the pro-peptide through thepromoter which controls expression of the zymogen or the mature protein.Alternatively, the two expressed genes would be combined in the samevector. In preferred embodiments of this application, the pro-peptideemployed is cleaved under physiological conditions.

In another aspect the present invention provides a fusion proteincomprising (a) a pro-peptide derived from an autocatalytically maturingzymogen and (b) a polypeptide that is heterologous to the pro-peptide.In one embodiment, the polypeptide is a therapeutic or nutritionalpeptide or protein which can be administered as an inactive fusionprotein. Activation or maturation through cleavage would only occur uponits delivery at the unique physiological conditions prevalent at thetarget organ, tissue or bodily fluid for example in the mammalianstomach, gut, kidneys, milk or blood. Cleavage might be enhanced by aprotease specific for the peptide, preferably the mature zymogenhomologous to the pro-peptide is used. This method is particularlyuseful for the delivery of orally ingested vaccines, cytokines, gastriclipase, peptide antibiotics, lactase and cattle feed enzymes whichfacilitate digestion, such as xylanase and cellulase. For example, atherapeutic or nutritional peptide or protein fused downstream of thechymosin pro-peptide might be activated in the mammal stomach uponingestion. The mature form of chymosin or the inactive precursor form ofchymosin may be added to assist in the cleavage of the nutritional ortherapeutic peptide.

Accordingly, in one embodiment the present invention provides apharmaceutical composition comprising a fusion protein which comprises(a) a pro-peptide derived from an autocatalytically maturing zymogen and(b) a polypeptide that is heterologous to the pro-peptide in admixturewith a suitable diluent or carrier. The composition may be administeredorally, intravenously or via any other delivery route.

The fusion protein and/or mature protein may also be produced in anedible food source, such as animal milk or in an edible crop, which maybe consumed without a need for further purification. Accordingly, inanother embodiment the present invention provides a food compositioncomprising a fusion protein which comprises (a) a pro-peptide derivedfrom an autocatalytically maturing zymogen and (b) a polypeptide that isheterologous to the pro-peptide in admixture with a suitable diluent orcarrier. The nutritional composition may be mixed with any liquid orsolid food and consumed by a human or animal.

The compositions of the invention may include the chimeric nucleic acidsequences or an expression vector containing the chimeric nucleic acidsequences of the present invention. In such an embodiment, the fusionprotein is produced in vivo in the host animal. The chimeric nucleicacid sequences of the invention may be directly introduced into cells ortissues in vivo using delivery vehicles such as retroviral vectors,adenoviral vectors and DNA virus vectors. The chimeric nucleic acidsequences may also be introduced into cells in vitro using physicaltechniques such as microinjection and electroporation or chemicalmethods such as co-precipitation and incorporation of nucleic acid intoliposomes. Expression vectors may also be delivered in the form of anaerosol or by lavage.

The present invention is also useful in the purification process ofrecombinant proteins. In one embodiment, a cell extract containing anexpressed pro-peptide-heterologous fusion protein is applied to achromatographic column. Selective binding of the fusion protein toantibodies raised against the pro-peptide sequence and immobilized ontothe column, results in selective retention of the fusion protein.Instead of relying on antibodies against the pro-peptide sequence, agene encoding another immunogenic domain or a gene encoding a peptidewith affinity for a commonly used column material, such as cellulose,glutathione-S-transferase or chitin, or any other desirable tag, may beincluded in the gene fusion.

In another envisaged application, a peptide encoding a sequence whichresults in anchoring of the fusion protein in the cell wall would beincluded in the construct. Suitable anchoring proteins for thisapplication would be yeast α-gluttenin FLO1, the Major Cell Wall Proteinof lower eukaryotes, and a proteinase of lactic acid bacteria (PCT94/18330) Expression of a fusion protein would result in immobilizationof the protein of interest to cell wall. The protein of interest couldbe isolated by washing the cells with water or washing buffer. Uponcleavage the cells could be removed using a simple centrifugation stepand the protein could be isolated from the washing buffer.

The following non-limiting examples are illustrative of the presentinvention.

EXAMPLES Example 1

In the first example, the protein hirudin was prepared as a fusionprotein with the chymosin pro-peptide and hirudin was shown to be activein cellular extracts of E. coli upon performance of a cleavage reaction.

Construction of a pGEX-Pro-Hirudin Fusion.

The fusion protein that we studied comprises the pro-peptide of calfchymosin B (Foltmann et al, 1977; Harris et al., 1982, Nucl. Acids.Res., 10: 2177-2187) fused to hirudin variant 1 (Dodt et al., 1984, FEBSLetters 65: 180-183). The hybrid gene which encoded this fusion proteinwas constructed using standard PCR methods (Horton et al., 1989, Gene,77: 61-68). The DNA sequence for this Pro-Hirudin fusion was cloned intopGEX-4T-3 (Pharmacia), downstream of the gene encodingglutathion-S-transferase (GST). The complete sequence of theGST-Pro-Hirudin sequence is shown in FIG. 1.

Growth of E. coli Transformed with pGEX-4T-3 and pGEX-Pro-Hirudin.

Plasmids pGEX-4T-3 and pGEX-Pro-Hirudin were transformed into E. coli.strain DH5α to allow for high level of expression. A single colony wasused to inoculate 5 ml LB-amp broth. These cultures were grownovernight. One ml of each overnight culture was used to inoculate 50 mlof LB-amp broth. These cultures were grown until OD₆₀₀=0.6. At this OD,IPTG (final concentration 1 mM) was added to induce the expression ofthe GST and GST-Pro-Hirudin fusion proteins. After this induction, thecultures were grown for an additional 3 hours at 37° C. The cells werepelleted at 5000×g for 10 minutes, and resuspended in 5 ml Tris BufferedSaline (TBS). The resuspended cells were sonicated and centrifuged at12000×g for 15 minutes to separate the inclusion bodies (pelletfraction) from the soluble proteins (supernatant fraction). Westernblotting of both the pellet and supernation fraction indicated thatunder the growing conditions described above, significant amounts(5-10%) of the GST and GST-Pro-Hirudin protein were found in thesupernatant fraction. The rest (90-95%) accumulated in inclusion bodies(results not shown).

Hirudin Activity Measurements

The supernatant fractions of both the GST and GST-Pro-Hirudin weretested for anti-thrombin activity. The samples were treated as follows:A) 201 supernatant+20 μl water B) 20 μl supernatant+20 μl of 100 mMSodium Phosphate pH 2.0 C) 20 μl supernatant+20 μl of 100 mM SodiumPhosphate pH 2.0+2 μg chymosin (Sigma) D) 20 μl supernatant+20 μl of 100mM Sodium Phosphate pH 4.5E) 20 μl supernatant+20 μl of 100 mM SodiumPhosphate pH 4.5+2 μg chymosin. These samples were incubated at roomtemperature for 1 hour. A total of 10 μl of the samples was added to 1ml assay buffer (20 mM Tris [pH 7.5], 100 mM NaCl, 5 mM CaCl₂, 0.1 unitof thrombin) and incubated for 2-3 minutes before the addition of 50 μlp-tos-gly-pro-arg-nitroanilide (1 mM). Thrombin activity was measured asa function of chromozyme cleavage by monitoring the increase inabsorption at 405 nm over time (Chang, 1983, FEBS Letters, 164:307-313). The AAbs (405 nm) was determined after 2 minutes. The resultof the activity measurements are indicated in Table 1.

As can be seen from Table 1, the only extract which exhibitedsignificant anti-thrombin activity was the extract containing theGST-Pro-Hirudin fusion which was treated at pH 4.5 and supplemented with2 μg chymosin (E). Western blotting (results not shown) indicated thatapart from treatment at pH 4.5, complete cleavage was also observed whenthe GST-Pro-Hirudin fusion which was treated at pH 2.0 and supplementedwith 2 μg chymosin. It has been well documented that unprocessedchymosin when exposed at pH 2.0, forms a pseudochymosin, before itmatures into chymosin (Foltmann et al., 1977, Scand. J. Clin. Lab.Invest. 42: 65-79; Foltmann, 1992, Proc. Natl. Acad. Sci. 74: 2321-2324;McCaman and Cummings, 1988, J. Biol. Chem. 261: 15345-15348) The pseudochymosin cleavage site is located between the Phe²⁷-Leu²⁸ peptide bondand is indicated in FIG. 1. The inability of the GST-Pro-Hirudin fusion,which was treated at pH 2.0 and supplemented with 2 μg chymosin, toinhibit thrombin activity might be explained by the fact that cleavageoccurred at the Phe²⁷-Leu²⁸ peptide bond rather than at the Phe⁴³-Val⁴⁴peptide bond which separates the chymosin pro-peptide from the maturehirudin. It has been well documented that (Loison et al., 1988,Bio/Technology, 6: 72-77) mature hirudin is only active when it does nothave any additional amino acids attached to its native N-terminalsequence.

Example 2

In the second example, the protein carp growth hormone (cGH) wasprepared as a fusion of pro-chymosin. Carp growth hormone was shown tobe present in cellular extracts of E. coli upon performance of thecleavage reaction.

Construction of a pHis-Pro-cGH Fusion

A fusion protein was constructed which comprises the pro-peptide of calfchymosin B (Foltmann et al., (1977), Harris et al., 1982, Nucl. AcidsRes. 10: 2177-2187 fused to carp growth hormone (Koren et al. (1989),Gene 67: 309-315). The hybrid gene which encoded this fusion protein wasconstructed using PCR mediated gene-fusion. The DNA sequence for thisPro-cGH fusion was cloned into pUC19 yielding plasmid pPro-cGH. ThePro-cGH gene fusion was released from pPro-cGH by SwaI/KpnI digestionand inserted into the PvuII/KpnI site of pRSETB (Invitrogen Corp.),containing a poly-histidine tag, facilitating purification, and anenterokinase recognition and cleavage site to generate pHis-Pro-cGH. Thecomplete sequence of the His-Pro-cGH insert is shown in FIG. 2.

Growth of E. coli Transformed with pHis-Pro-cGH

Plasmid pHis-Pro-cGH was transformed into E. coli BL21 strain to allowfor high levels of expression. A single colony was used to inoculateLB-amp broth These cultures were grown overnight. One ml of each o/nculture was used to inoculate 50 ml of LB-amp broth. These cultures weregrown until OD₆₀₀=0.6. At this OD, IPTG (final concentration 0.5 mM) wasadded to induce the expression of the His-Pro-cGH fusion protein. Afterthis induction, the cultures were grown for an additional 3 hours at 37°C. The cells were pelleted at 5000×g for 10 minutes, and resuspended in5 ml PBS (pH 7.3) buffer. The resuspended cells were disrupted by aFrench-Press and centrifuged at 10,000×g for 10 minutes. Inclusionbodies were resuspended in 5 ml of water and dissolved by slow additionof NaOH. 1 ml of 10×PBS was added to this solution and the volume wasadjusted to 10 ml. The pH of the solution was adjusted to 8.0 by slowaddition of HCl and the solution was incubated at 4° C. for 2 hours. ThepH was adjusted to 7.5 and at this point the solution was centrifuged at10,000 g for 15 minutes to remove insolubles. The fusion protein wasthen purified by chelating affinity chromatography using Hi-Trap metalbinding columns (Pharmacia). The column was saturated with Zn⁺⁺ ions andthen used to affinity purify His-Pro-cGH fusion protein in accordancewith the instructions provided by the manufacturer.

Cleavage of cGH Produced in E. coli Transformed with pHis-Pro-cGH

In order to cleave the fusion protein 15 μl (ca 1 μg) of the proteinprep was treated with either 17 μl of PBS (Uncut), 14 μl of PBS and 3 μlof enterokinase (Cut (EK)), or 16 μl of phosphate buffer (pH 2) and 1 μlof chymosin (Cut (PRO)). All samples were incubated at 37° C. for 2hours and then analysed by SDS-PAGE followed by western blotting. Theprimary antibody used was a rabbit anti-serum prepared against cGH. Thesecondary antibody was goat anti-rabbit IgG which was conjugated withalkaline phosphatase.

As can be seen from FIG. 3, cleavage of the fusion protein was observedwith enterokinase yielding a protein band corresponding to thecalculated molecular mass of the Pro-cGH fusion (26 kDa). Similarly thecleavage with chymosin yielded a protein band corresponding to theexpected theoretical molecular mass of the cGH (approximately 22 kDa)polypeptide.

Example 3

In this example, the protein carp growth hormone (cGH) was prepared as afusion of pro-chymosin. The carp growth hormone fusion protein wascleaved with the gut extract from red turnip beetle, thus illustratingan in vivo application of the invention.

His-Pro-cGH was prepared following the protocol of example 2. Gutextract was prepared from larvae of the red turnip beetle as follows.Red turnip beetle eggs (Entomoscelis americana Brown (Coleoptera:Chrysomelidae), were laid by laboratory-reared adults and stored at −20°C. for at least three months before use. Eggs were hatched in dishescontaining moist filter paper, and larvae were maintained on canolaseedlings. Only larvae that were actively feeding were used. Midgutsfrom second instar larvae were removed by dissection in saline solutionand stored in saline at −20° C. Guts were thawed, rinsed in ddH₂O (50 μlper gut). The homgenate was centrifuged at 16,000×g (10 min, 4° C.) andthe decanted supernatant was used in the proteolyic assay.

As can be observed in FIG. 4, extracts prepared from the gut of redturnip beetle cleaved the fusion protein and released the cGHpolypeptide. Cleavage was not observed to be complete. This could be dueto the fact that the pH in the gut extract was not optimal for thecleavage reaction to proceed.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is intended to cover various modificationsand equivalent arrangements included within the spirit and scope of theappended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

DETAILED FIGURE LEGENDS

FIG. 1. The nucleic acid and deduced amino acid sequence of aGST-Pro-Hirudin sequence. The deduced sequence of the chymosinpro-peptide has been underlined and the deduced hirudin protein sequencehas been italicized. The hirudin nucleic acid sequence was optimized forplant codon usage. The pseudochymosin cleavage site between Phe27-Leu28and the peptide bond separating the pro-chymosin and mature hirudin (Phe42-Val43) are indicated with an arrow (↑).

FIG. 2. The nucleic acid and deduced amino acid sequence of aHis-Pro-cGH sequence. The deduced sequence of the chymosin pro-peptidehas been underlined and the deduced amino acid of cGH has beenitalicized. The cleavage site of enterokinase between (Lys31-Asp32) andthe peptide bond separating the pro-chymosin and the mature cGH(Phe84-Ser85) are indicated with an arrow (↑). The poly-histidine site(His5-His10) and the enterokinase recognition site (Asp27-Lys31) arealso indicated.

FIG. 3 is a schematic diagram of the His-Pro-cGH fusion construct. Theenterokinase cleavage site (enterokinase cleavage) and pro-chymosincleavage site (PRO Cleavage) are indicated with an arrow (↑).

FIG. 4 illustrates the cleavage of purified His-Pro-cGH. Shown on theWestern blot probed with an anti cGH antibodies are column purifiedHis-Pro-cGH protein extracts from E. coli cells expressing theHis-Pro-cGH fusion construct treated with enterokinase (Cut (EK)),mature chymosin at low pH (Cut (PRO)) and the control which was treatedwith PBS buffer (Uncut).

FIG. 5 illustrates the cleavage of purified His-Pro-cGH. Shown on theWestern blot probed with anti cGH antibodies are column purifiedHis-Pro-cGH protein extracts from E. coli cells expressing theHis-Pro-cGh fusion construct treated with mature chymosin at low pH (Cut(PRO)), treated with enterokinase (Cut (EK)), treated with gut extractfrom red turnip beetle (Cut (Red Turnip Gut)).

TABLE 1 Activity measurements of bacterial extracts containing GST(Glutathion-S-transferase) and GST-Pro-Hirudin fusions. Δ Abs (405 Δ Abs(405 nm)/2 min nm)/2 min Sample [Test 1] [Test 2] 1 unit Thrombin 0.0880.066 A: GST 0.087 0.082 B: GST pH 2.0 0.082 0.073 C: GST pH 2.0 + 2 μgchymosin 0.063 0.073 D: GST pH 4.5 0.087 0.086 E: GST pH 4.5 + 2 μgchymosin 0.087 0.087 A: GST-PRO-HIR 0.076 0.071 B: GST-PRO-HIR pH 2.00.072 0.064 C: GST-PRO-HIR pH 2.0 + 2 μg 0.066 0.070 chymosin D:GST-PRO-HIR pH 4.5 0.078 0.075 E: GST-PROHIR pH 4.5 + 2 μg chymosin0.0002 0.0001 Hirudin 2 μg 0.0001 0.0001

1. A method for the preparation of a recombinant polypeptide comprisinga) transforming a non-human host cell with an expression vectorcomprising: (1) a nucleic acid sequence capable of regulatingtranscription in a host cell, operatively linked to (2) a chimericnucleic acid sequence that encodes a fusion protein, wherein saidchimeric nucleic acid sequence comprises (a) a nucleic acid sequenceencoding a full-length chymosin pro-peptide, linked in reading frame to(b) a nucleic acid sequence that is heterologous to the pro-peptide andthat encodes the recombinant polypeptide, wherein the heterologousnucleic acid sequence is located immediately downstream of the nucleicacid sequence encoding the chymosin pro-peptide; operatively linked to(3) a nucleic acid sequence encoding a termination region that isfunctional in said host cell, b) growing the non-human host cell toproduce said fusion protein, c) obtaining said fusion protein from saidnon-human host cell, and d) contacting said fusion protein with a matureform of an autocatalytically maturing aspartic protease that is capableof cleaving the chymosin pro-peptide, whereby said chymosin pro-peptideis cleaved from said fusion protein to release said recombinantpolypeptide.
 2. The method according to claim 1 wherein said asparticprotease of step d) is selected from the group consisting of chymosin,pepsin, pepsinogen, cathepsin and yeast proteinase A.
 3. The methodaccording to claim 1 wherein the recombinant polypeptide is hirudin orcarp growth hormone.
 4. The method according to claim 1 wherein thechimeric nucleic acid sequence does not include a sequence encoding amature form of chymosin.
 5. The method according to claim 1, whereinstep d) is effected at a pH of from about 2 to about 4.5.
 6. The methodaccording to claim 1 wherein step d) is effected in vitro.
 7. The methodaccording to claim 1 wherein step d) is effected in vivo.
 8. The methodaccording to claim 7 wherein step d) is effected in the milk, thestomach, or the gut of an animal.
 9. The method according to claim 1wherein the aspartic protease of step d) is chymosin.
 10. The methodaccording to claim 1 wherein the aspartic protease of step d) isheterologous to the chymosin pro-peptide.
 11. The method according toclaim 9 wherein step d) is effected in vitro.
 12. The method accordingto claim 9 wherein step d) is effected in vivo.
 13. The method accordingto claim 12 wherein step d) is effected in the stomach, gut, or milk ofan animal.
 14. The method according to claim 1 wherein said nucleic acidsequences are deoxyribonucleic acid (DNA) sequences.
 15. The methodaccording to claim 1 wherein said aspartic protease of step d) ispepsin.
 16. A method for the preparation of a recombinant polypeptide,comprising a) transforming a host cell with an expression vectorcomprising: (1) a nucleic acid sequence capable of regulatingtranscription in a host cell, operatively linked to (2) a chimericnucleic acid sequence that encodes a fusion protein, wherein saidchimeric nucleic acid sequence comprises (a) a nucleic acid sequenceencoding a full length chymosin pro-peptide, linked in reading frame to(b) a nucleic acid sequence that is heterologous to the pro-peptide andthat encodes the recombinant polypeptide, wherein the heterologousnucleic acid sequence is located immediately downstream of the nucleicacid sequence encoding the chymosin pro-peptide; operatively linked to(3) a nucleic acid sequence encoding a termination region that isfunctional in said host cell, wherein the host cell is selected from thegroup consisting of bacterial cells, yeast cells and plant cells, b)growing the host cell to produce said fusion protein; c) contacting saidfusion protein in vivo with a mature form of an autocatalyticallymaturing aspartic protease that cleaves the pro-peptide by expressingsaid autocatalytically maturing aspartic protease in said host cell,whereby said pro-peptide is cleaved from said fusion protein to releasesaid recombinant polypeptide.